In vitro method for detecting intestinal barrier failure in animals by determining ovotransferrin

ABSTRACT

The present invention pertains to an in vitro method for detecting intestinal barrier failure in animals, the method comprising the following steps: a) collecting intestinal sample material of an individual animal or of an animal population; and b) determining the amount of at least one protein marker contained in said sample material; wherein the at least one protein marker comprises or consists of ovotransferrin or a functional fragment thereof, and wherein an increased amount of said at least one protein marker contained in said sample versus a reference sample indicates intestinal barrier failure.

FIELD OF THE INVENTION

The present invention relates to an in vitro method for detectingintestinal barrier failure in animals. More specifically, the presentinvention pertains to an acute phase protein (APP)-based method forevaluating the gut health status of an individual animal and of ananimal population, respectively.

BACKGROUND OF THE INVENTION

Intestinal health is critically important for the welfare andperformance of livestock animals. Enteric diseases that affect thestructural integrity of the gastrointestinal tract (GIT) lead to higheconomic losses due to reduced weight gain, poor feed conversionefficiency, increased mortality rates and greater medication costs(M'Sadeq, S. A., Wu, S., Swick, R. A. & Choct, M. (2015). Towards thecontrol of necrotic enteritis in broiler chickens with in-feedantibiotics phasing-out worldwide. Animal Nutrition, 1, 1-11;Timbermont, L., Haesebrouck, F., Ducatelle, R. & Van Immerseel, F.(2011). Necrotic enteritis in broilers: an updated review on thepathogenesis. Avian Pathol, 40, 341-347).

An intact intestinal barrier provides a number of physiological andfunctional features, including nutrient digestion and absorption, hostmetabolism and energy generation, a stable microbiome, mucus layerdevelopment, barrier function, and mucosal immune responses (Kogut, M.H. and R. J. Arsenault (2016). Editorial: Gut health: The new paradigmin food animal production. Frontiers in Veterinary Science 3 (AUG)). Asthe largest organ in the body, the gut serves as a selective barrier totake up nutrients and fluids into the body, while excluding undesirablemolecules and pathogens. Therefore, proper gut barrier function isessential to maintain optimal health and balance throughout the body,and represents a key line of defense against foreign antigens from theenvironment.

Coccidiosis and necrotic enteritis (NE) probably are the most commonenteric diseases of poultry (Dalloul, R. A. & Lillehoj, H. S. (2006).Poultry coccidiosis: recent advancements in control measures and vaccinedevelopment. Expert Rev Vaccines, 5, 143-163; Williams, R. B. (2005).Intercurrent coccidiosis and necrotic enteritis of chickens: rational,integrated disease management by maintenance of gut integrity. AvianPathol, 34, 159-180). In poultry, coccidiosis can be caused by multiplespecies belonging to the genus Eimeria, from which Eimeria acervulina,E. maxima and E. tenella are the most common species in intensivelyreared broilers. Depending on the species, the lesions can range from alimited malabsorptive enteritis (E. acervulina) to more severeinflammation of the intestinal wall (E. maxima) and even extensivecaecal haemorrhage and death (E. tenella) (Chapman, H. D. (2014).Milestones in avian coccidiosis research: a review. Poult Sci, 93,501-511). Furthermore, the presence of Eimeria species can alsoexacerbate the outcome of co-infection with bacterial pathogens such asClostridium perfringens (Moore, R. J. (2016). Necrotic enteritispredisposing factors in broiler chickens. Avian Pathol, 45, 275-281).Indeed, the mucosal damage caused by these coccidial pathogens is animportant predisposing factor for necrotic enteritis. NE is the mostcommon clostridial enteric disease in poultry, which typically occurs inbroiler chickens. The disease is caused by C. perfringens and can occureither as an acute clinical or as a mild subclinical form. Acute NEtypically leads to a massive increase in flock mortality. The morecommon subclinical form is characterized by multifocal necrosis andinflammation of the small intestine with a significant decline in growthperformance. The reduction in performance is not only associated withimpaired growth rate and feed conversion during production, but alsowith increased condemnation rates in broilers due to hepatitis atprocessing (Paiva, D. & McElroy, A. (2014). Necrotic enteritis:Applications for the poultry industry. Journal of Applied PoultryResearch, 23, 557-566). Both coccidiosis and necrotic enteritis can bepresent in a flock without showing clinical signs. Therefore, multiplebirds have to be sacrificed for macroscopic examination of the intestineto diagnose the disease.

Similar considerations apply for other enteric diseases or conditions inlivestock animals which are leading to mucosal damage, such as severebacterial overgrowth in the small intestine, all forms of excessive gutinflammation, exposure to mycotoxins, and every condition which leads tointestinal barrier failure. In addition, intestinal barrier failuremight enable normal inhabitants of the GIT, like Enterococcus caecorumto invade the systemic circulation. This can lead to further diseaseslike arthritis and osteomyelitis and finally lead to lower performanceof the animals or the animal flock, respectively.

A marker, or a set of markers, that can accurately detect intestinalbarrier failure or intestinal inflammation and concomitant perturbationof the intestinal integrity at an early stage would thus be highlydesirable.

Recently, there has been increased interest in research on intestinalpermeability in chickens, resulting in different strategies to measureintestinal inflammation and concomitant intestinal barrier failure.However, none of the proposed strategies represent a good marker for thebroiler industry as they are either not applicable under fieldconditions (e.g. oral administration of a marker that can be measured inthe blood on a later timepoint (Gilani, S., Howarth, G. S., Kitessa, S.M., Tran, C. D., Forder, R. E. A. & Hughes, R. J. (2017). New biomarkersfor increased intestinal permeability induced by dextran sodium sulphateand fasting in chickens. J Anim Physiol Anim Nutr (Berl), 101,e237-e245; Vicuna, E. A., Kuttappan, V. A., Tellez, G.,Hernandez-Velasco, X., Seeber-Galarza, R., Latorre, J. D., et al.(2015). Dose titration of FITC-D for optimal measurement of entericinflammation in broiler chicks. Poult Sci, 94, 1353-1359) ornon-specific for intestinal barrier failure, such as serum markers thatcan be elevated by non-gastrointestinal conditions as well (Chen, J.,Tellez, G., Richards, J. D. & Escobar, J. (2015). Identification ofPotential Biomarkers for Gut Barrier Failure in Broiler Chickens. FrontVet Sci, 2, 14; O'Reilly, E. L. & Eckersall, P. D. (2014). Acute phaseproteins: a review of their function, behaviour and measurement inchickens. Worlds Poultry Science Journal, 70, 27-43; Xie, H., Newberry,L., Clark, F. D., Huff, W. E., Huff, G. R., Balog, J. M., et al. (2002).Changes in serum ovotransferrin levels in chickens with experimentallyinduced inflammation and diseases. Avian Dis, 46, 122-131).

It was thus a remaining need to provide a fast and reliable, ideallynon-invasive ante mortem method for determining whether or not anindividual animal or an animal population suffers from intestinalbarrier failure that can be performed under field conditions at low costand with minimal effort.

SUMMARY OF THE INVENTION

Accordingly, one objective of the present invention is to provide an invitro method for detecting intestinal barrier failure in animals, themethod comprising the following steps:

-   -   a) collecting intestinal sample material of an individual animal        or of an animal population; and    -   b) determining the amount of at least one protein marker        contained in said sample material;        wherein

-   the at least one protein marker comprises or consists of    ovotransferrin or a functional fragment thereof,

-   and wherein

-   an increased amount of said at least one protein marker contained in    said sample versus a reference sample indicates intestinal barrier    failure.

An additional aspect of the present application is the use ofovotransferrin or of functional fragments thereof as intestinal markersfor detecting intestinal barrier failure in an animal subject or in ananimal population.

A further objective of the present invention is the provision of an invitro method for detecting the extent of intestinal barrier failure inanimals, the method comprising the following steps:

-   -   a) collecting intestinal sample material of a specific animal or        of an animal population; and    -   b) determining the amount of at least one protein marker        contained in the sample material;        wherein

-   the at least one protein marker comprises or consists of    ovotransferrin or a functional fragment thereof,

-   and wherein

-   the amount of said at least one protein marker contained in the    sample indicates the extent of the intestinal barrier failure.

Finally, the present invention provides an in vitro method formonitoring the status of the intestinal barrier in animals, the methodcomprising the following steps:

-   -   a) collecting intestinal sample material of a specific animal or        of an animal population at consecutive points in time;    -   b) determining the amount of at least one protein marker        contained in the samples obtained in step a); and    -   c) determining deviations in the amounts of said at least one        protein marker contained in the samples obtained in step a);        wherein

-   the at least one protein marker comprises or consists of    ovotransferrin or a functional fragment thereof.

In the following, the crucial aspects of the present invention aredescribed in detail.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have unexpectedly found that the amount of acutephase protein (APP)-based markers contained in sample material of animalorigin correlates with the manifestation of intestinal barrier failure.In particular, it was found that the amount of ovotransferrin, orfunctional fragments thereof, in intestinal sample material of animalorigin correlates with intestinal barrier failure.

More specifically, the inventors have found that an increase in theamount of ovotransferrin, or functional fragments thereof, contained inintestinal sample material of animal origin versus a reference sampleindicates intestinal barrier failure.

Accordingly, the present invention pertains to an in vitro method fordetecting intestinal barrier failure in animals, the method comprisingthe following steps:

-   -   a) collecting intestinal sample material of an individual animal        or of an animal population; and    -   b) determining the amount of at least one protein marker        contained in said sample material;        wherein

-   the at least one protein marker comprises or consists of    ovotransferrin or a functional fragment thereof,

-   and wherein

-   an increased amount of said at least one protein marker contained in    said sample versus a reference sample indicates intestinal barrier    failure.

As used in the context of the present invention, the term “intestinalbarrier failure” refers to conditions in which the intestinal barrierfunction is significantly impaired (e.g. due to oxidative stress, poorlydigestible protein, coccidiosis, etc.); and comprises conditions ofintestinal barrier dysfunction, intestinal leakages/permeability andconditions caused by histopathologic injuries. Intestinal barrierfailure is often associated with inflammatory processes. The terms“intestinal barrier failure” and “gut barrier failure” may be usedinterchangeably.

The reference sample is a species-specific control representing anintact intestinal barrier or gut barrier. Suitable reference samples aresamples obtained from an individual animal or from an animal populationof the same animal species or sub-species, whereby said animal or saidanimal population has a proven intact intestinal barrier.

As an example, a reference sample may be taken within an animal trialfrom an animal of a non-treated control, which was checked viapathology, histopathology and/or other measures to have no signs ofintestinal barrier failure.

The ovotransferrin markers may be detected and quantified using thecommonly known, conventional techniques, such as immunoassays like ELISA(Enzyme-linked Immunosorbent Assay), lateral flow assays, massspectrometry (MS) analyses, or any method enabling the detection ofproteins or functional fragments thereof.

In one specific embodiment, the ovotransferrin, or functional fragmentsthereof, is detected and quantified via ELISA.

The use of monoclonal antibodies enables a specific detection in thecomplex sample matrix used for the analysis.

The method of the present invention may be used for determining whetheror not an individual animal suffers from intestinal barrier failure. Inthat case, the intestinal sample material originates from an individualanimal.

The individual animal may for example be a pet or domestic animal, afarm animal as occurring in live stocks, a wild-living animal or a zooanimal. Further, animal individuals being transported for slaughter orfor re-location may be examined using the above method.

In one embodiment, the individual animal is an avian subject.

The avian subject to be tested is preferably poultry. Preferred poultryaccording to the invention are chickens, turkeys, ducks and geese. Thepoultry can be optimized for producing young stock. This type of poultryis also referred to as parent and grandparent animals. Preferred parentand grandparent animals are, accordingly, (grand)parent broilers,(grand)parent ducks, (grand)parent turkeys and (grand)parent geese.

The poultry according to the invention can also be selected from fancypoultry and wild fowl. Preferred fancy poultry or wild fowl arepeacocks, pheasants, partridges, guinea fowl, quails, capercailzies,goose, pigeons and swans. Further preferred poultry according to theinvention are ostriches and parrots. Most preferred poultry according tothe invention are broilers.

The intestinal sample material obtained from an individual animal may beselected from the group consisting of gut content samples, samples ofbodily excrements and solutions or suspensions thereof; and frommaterials being contaminated with bodily excrements. The term “gutcontent” is to be understood as the content of the small intestine, thecontent of the large intestine and/or the content of the caecum. Methodsfor taking such gut content samples are known in the art.

As used in the context of the present invention, bodily excrements arefecal or cecal excrements. Materials being contaminated with bodilyexcrements are, for example, dust samples, swab samples, litter samples,liquid manure samples, fur samples, feather samples and skin samples.

In general, the term “litter” is to be understood as a mixture of animalexcrements with the bedding material.

As used in the context of this embodiment, the term “litter samples”refers to excremental droppings from an individual animal. Further, inthe context this embodiment, the term “liquid manure samples” refers toan excremental sample containing feces and urine from an individualanimal.

Samples from individual animals can be taken either directly from theanimal, e.g. with swabs. Alternatively and especially in case ofsingle-housed animals, the sample material can be collected from thefloor of the pen, cage or slat. The sample material has to be assignableto the investigated animal.

In one embodiment, the intestinal sample material used for determiningwhether or not an individual animal suffers from intestinal barrierfailure is feces.

For specific applications, it is also useful to analyze gut contentsamples, e.g. samples from the small intestine, samples from the largeintestine and/or samples from the caecum.

Suitable sample volumes are, for example, 0.05 ml to 20 ml or 0.1 to 20ml, in particular 0.2 to 10 ml, preferably 0.5 to 5 ml. Suitable samplemasses are, for example 0.05 g to 20 g or 0.1 to 20 g, in particular 0.2to 10 g, preferably 0.5 to 5 g

In an alternative embodiment, the method is used for determining whetheror not an animal population suffers from intestinal barrier failure. Inthat case, the sample material originates from the group of animals tobe tested.

As used herein, the term “animal population” refers to a group of animalindividuals belonging to the same species. The animal population may forexample be a group of pets or domestic animals as occurring in animalbreeding, a group of farm animals as occurring in livestock productionor in livestock breeding, or a group of wild-living animals or zooanimals.

In one embodiment, the animal population is an animal flock as occurringin livestock production processes. For example, the animal population orthe animal flock can be an avian flock; a flock of sheep, goat orcattle, a flock of horses or a flock of pigs.

In one specific embodiment, the animal population is an avianpopulation.

The animal population preferably is an avian flock. The avian flockaccording to the invention is preferably poultry. Preferred poultryaccording to the invention are chickens, turkeys, ducks and geese. Thepoultry can be optimized for producing young stock. This type of poultryis also referred to as parent and grandparent animals. Preferred parentand grandparent animals are, accordingly, (grand)parent broilers,(grand)parent ducks, (grand)parent turkeys and (grand)parent geese.

The poultry according to the invention can also be selected from fancypoultry and wild fowl. Preferred fancy poultry or wild fowl arepeacocks, pheasants, partridges, guinea fowl, quails, capercailzies,goose, pigeons and swans. Further preferred poultry according to theinvention are ostriches and parrots. Most preferred poultry according tothe invention are broilers.

The method of the present invention is particularly suitable fordetermining the health status of an animal population via bulk testing.As used herein, the term “bulk testing” refers to a test method, whereinthe sample material is a pooled sample of an animal population. A“pooled sample” in the context of this embodiment is to be understood asa composite sample from randomly selected separate samples, one sampletaken with one or several moistened fabric swabs or pooled samples madeup of separate samples of fresh samples taken at random from a number ofsites in the house or space in which the animal population or the animalflock is kept. It may be necessary that the sample material ishomogenized prior to sample analysis. Suitable homogenization techniquesare known in the art.

The pooled samples reflect the amount of ovotransferrin present in theanimal population.

The sample material obtained from an individual animal may be selectedfrom the group consisting of gut content samples, samples of bodilyexcrements and solutions or suspensions thereof; and from materialsbeing contaminated with bodily excrements. Materials being contaminatedwith bodily excrements are, for example, dust samples, swab samples,litter samples, liquid manure samples, fur samples, feather samples andskin samples.

As used in the context of this embodiment, the term “litter samples”refers to mixed excremental droppings in the pen, cage or slat. Further,in the context this embodiment, the term “liquid manure samples” refersto mixed excremental samples containing feces and urine.

These litter samples can, for example, be collected from an animalpopulation using the overshoe method or using litter grabs at differentplaces in the pen.

Boot swabs being sufficiently absorptive to soak up moisture areparticularly suitable for collecting pooled animal samples. Tube gauzesocks are also acceptable.

In case the animal population is kept in cages or slats, the excrementalsamples may be collected by a conveying belt.

In one embodiment, the sample material used for determining whether ornot an animal population suffers from intestinal barrier failure isfeces. Preferably, the sample material is a pooled fecal sample derivingfrom an avian flock.

For specific applications, it is also useful to analyze pooled gutcontent samples, e.g. pooled samples from the small intestine, pooledsamples from the large intestine and/or pooled samples from the caecum.

Suitable sample volumes are, for example, 0.1 to 20 ml, in particular0.2 to 10 ml, preferably 0.5 to 5 ml. Suitable sample masses are, forexample 0.1 to 20 g, in particular 0.2 to 10 g, preferably 0.5 to 5 g.

Depending on the sample material and storage conditions, it may behelpful to stabilize the samples taken in order to avoid enzymaticdegradation of the ovotransferrin contained in the samples, for exampleby treating the samples with protease inhibitors. Preferably, thestabilizing agent is added to the sample immediately after samplecollection.

In accordance with the above, one specific embodiment of the presentapplication pertains to an in vitro method for detecting intestinalbarrier failure in an avian flock, the method comprising the followingsteps:

-   -   a) collecting and pooling fecal sample material deriving from        said avian flock;    -   b) optionally stabilizing the pooled sample material; and    -   c) determining the amount of ovotransferrin contained in said        pooled sample material;        wherein

-   an increased amount of ovotransferrin contained in the sample versus    a reference sample indicates intestinal barrier failure.

In addition to the above, the inventors have unexpectedly found that theamount of ovotransferrin contained in intestinal sample materialderiving from an individual animal or from an animal populationcorrelates with the extent of intestinal barrier failure. Accordingly,the present invention provides an in vitro method for detecting theextent of intestinal barrier failure in animals, the method comprisingthe following steps:

-   -   a) collecting intestinal sample material of a specific animal or        of an animal population; and    -   b) determining the amount of at least one protein marker        contained in the sample material;        wherein

-   the at least one protein marker comprises or consists of    ovotransferrin or a functional fragment thereof,

-   and wherein

-   the amount of said at least one protein marker contained in the    sample indicates the extent of the intestinal barrier failure.

Suitable sample materials testing parameters and—conditions are asdefined above.

In a particularly preferred embodiment, the intestinal sample materialis a pooled fecal sample deriving from an avian flock and the at leastone protein marker is ovotransferrin or a functional fragment thereof.

The present invention provides the abovementioned methods for detectingintestinal barrier failure and for determining the extent thereof,respectively. This enables the farmer to make a qualified decision onwhether or not measures for improving intestinal health are to be taken.

Measures against the development and/or against the progression ofintestinal barrier failure involve feeding or administeringhealth-promoting substances, such as zootechnical feed additives, ortherapeutic agents. The term “administering” or related terms includesoral administration. Oral administration may be via drinking water, oralgavage, aerosol spray or animal feed. The term “zootechnical feedadditive” refers to any additive used to affect favorably theperformance of animals in good health or used to affect favorably theenvironment. Examples for zootechnical feed additives are digestibilityenhancers, i.e. substances which, when fed to animals, increase thedigestibility of the diet, through action on target feed materials; gutflora stabilizers; micro-organisms or other chemically definedsubstances, which, when fed to animals, have a positive effect on thegut flora; or substances which favorably affect the environment.Preferably, the health-promoting substances are selected from the groupconsisting of probiotic agents, praebiotic agents, botanicals,organic/fatty acids, zeolithes, bacteriophages and bacteriolytic enzymesor any combinations thereof.

The inventors have found that the testing procedures underlying thepresent invention may also be used for monitoring the intestinal healthstatus in animals.

As used in the context of this embodiment, the term “intestinal healthstatus” refers to status of the intestinal barrier.

By the above method, the development or the progression of an intestinalbarrier failure may be detected. On the other hand, the effectiveness ofmeasures taken against the development and/or against the progression ofintestinal barrier failure may be controlled.

Accordingly, the present invention also pertains to an in vitro methodfor monitoring the status of the intestinal barrier in animals, themethod comprising the following steps:

-   -   a) collecting intestinal sample material of a specific animal or        of an animal population at consecutive points in time;    -   b) determining the amount of at least one protein marker        contained in the samples obtained in step a); and    -   c) determining deviations in the amounts of said at least one        protein marker contained in the samples obtained in step a);        wherein

-   the at least one protein marker comprises or consists of    ovotransferrin or a functional fragment thereof.

Therein, an increase in the amount of ovotransferrin over time indicatesthe development or progression of intestinal barrier failure.Conversely, a decrease in the amount of ovotransferrin over timeindicates improvements in the intestinal health situation which may becaused by natural healing processes or by specific measures being takenagainst the development or progression of intestinal barrier failure.

An “increase” or a “decrease” in the amount of ovotransferrin typicallyrefers to a statistically relevant amount.

Suitable sample materials and testing parameters and—conditions are asdefined above. In a specific embodiment, the intestinal sample materialis a pooled sample deriving from an avian flock and the at least oneprotein marker is ovotransferrin or a functional fragment thereof.

As an example, after initial determination of the amount ofovotransferrin in an intestinal sample, the amount of ovotransferrin maybe monitored in test samples collected and analyzed in a weekly, dailyour hourly manner. In one embodiment, excremental samples are collectedand analyzed at consecutive days. The excremental test samples may betaken and analyzed on a daily basis from birth to slaughter.

In a specific embodiment for poultry, a first test sample is preferablytaken and analyzed during the initial growth phase (starter phase, day 5to day 10), a second test sample is taken and analyzed during theenhanced growth phase (day 11 to day 18) and, optionally, a third testsample is taken and analyzed on a later stage.

In an alternative embodiment, a first test sample is taken and analyzedin the initial growth phase and further test samples are taken andanalyzed for example on a daily basis during the enhanced growth phase,optionally until slaughter.

A further aspect of the present invention is the use of ovotransferrin,or functional fragments thereof, as intestinal markers for detectingintestinal barrier failure in an animal subject or in an animalpopulation. A specific embodiment of the present invention is the use ofovotransferrin as a fecal marker for detecting intestinal barrierfailure in an avian subject or in an avian population.

Applications of the methods according to the invention are for example(i) aiding in the diagnosis and/or prognosis of intestinal barrierfailure caused by enteric diseases; (ii) monitoring the progress orreoccurrence of intestinal barrier failure or (iii) aiding in theevaluation of treatment efficacy for an animal population undergoing orcontemplating treatment.

Applications of the invention in particular help to avoid loss in animalperformance like weight gain and feed conversion.

In the following, the invention is illustrated by non-limiting examplesand exemplifying embodiments.

EXAMPLES

Coccidiosis and necrotic enteritis in broiler chickens were used asmodels for intestinal barrier failure. Ovotransferrin serves as proteinmarker.

Necrotic Enteritis Trials—Sample Collection

Groups of 27 one-day-old Ross 308 broiler chickens were fed a diet richin proteins and non-starch polysaccharides which predispose to thedevelopment of necrotic enteritis. The detailed diet composition wasdescribed by Gholamiandehkordi et al. (Gholamiandehkordi, A. R.,Timbermont, L., Lanckriet, A., Van Den Broeck, W., Pedersen, K., Dewulf,J., et al. (2007). Quantification of gut lesions in a subclinicalnecrotic enteritis model. Avian Pathol, 36, 375-382). Other predisposingfactors consist of the administration of Gumboro vaccine to induce mildimmunosuppression and a ten-fold dose of coccidiosis vaccine (eitherParacox-8 or Hipracox, depending on the trial) to induce predisposingintestinal damage. To induce necrotic lesions, animals were challengedwith approximately 4.10⁸ CFU of the netB-positive C. perfringens strainCP56 on three consecutive days, after which the animals were euthanized.At necropsy, lesion scoring in the small intestine (duodenum, jejunumand ileum) was performed as described by Keyburn et al. (Keyburn, A. L.,Sheedy, S. A., Ford, M. E., Williamson, M. M., Awad, M. M., Rood, J. I.,et al. (2006). Alpha-toxin of Clostridium perfringens is not anessential virulence factor in necrotic enteritis in chickens. InfectImmun, 74, 6496-6500) as follows: score 0=no lesions, score 1=congestedintestinal mucosa, score 2=focal necrosis or ulcerations (1-5 foci),score 3=focal necrosis or ulcerations (6-15 foci), score 4=focalnecrosis or ulcerations 16 foci), score 5=patches of necrosis of 2-3 cmlong, score 6=diffuse necrosis. Birds with a lesion score of 2 or moreare classified as necrotic enteritis positive. Fresh cloacal sampleswere collected from all birds and frozen at −70° C. In addition, mixedlitter was collected from each pen and frozen at −70° C.

After lesion scoring, the samples were grouped according to the diseaseseverity of the animal, leading to the following disease severitygroups: birds that received all predisposing factors but were notchallenged with C. perfringens: negative control; birds challenged withC. perfringens but no necrosis: score 0 or challenged with C.perfringens and various severity degrees: score 2 (mild), score 3-4(moderate) or score 5-6 (severe).

Coccidiosis Trials—Sample Collection

Fifteen-day-old Ross 308 broiler chicks were orally challenged with E.acervulina and E. tenella. One mixed litter sample from each pen andcloacal samples from all birds were collected 7 days after challenge,when the chickens were euthanized for lesion scoring using the method ofJohnson and Reid (Johnson, J. & Reid, W. M. (1970). Anticoccidial drugs:lesion scoring techniques in battery and floor-pen experiments withchickens. Exp Parasitol, 28, 30-36). On the same day, mixed litter andcloacal content samples were collected from their age-matched controls.All challenged birds showed macroscopically visible lesions of Eimeriainfection, with a mean coccidiosis score of 5.11±0.51, whereas only oneout of then birds in the unchallenged control group was coccidiosispositive (coccidiosis score=1). All samples were stored at −70° C.

Ovotransferrin Detection by Enyzme-Linked Immunosorbent Assay (ELISA)

Eight samples from the negative control birds (not challenged with C.perfringens) and eight sample per necrosis score group from challengedbirds were selected. Also litter samples collected at the day ofnecropsy were included (one litter samples per pen, with in total 3samples from pens with non-challenged birds and 3 samples from pens withchallenged birds). Additionally, the ovotransferrin concentration wasdetermined in both cloacal samples and litter samples from thecoccidiosis trial. Therefore, 1 litter sample per pen was used, with intotal 5 samples from pens with non-challenged birds and 6 samples frompens with Eimeria-challenged birds. Additionally, 20 cloacal samplesfrom either Eimeria-challenged birds (n=10) or their non-challengedcontrols (n=10) were selected.

Unprocessed cloacal material or homogenized litter material was thawedat room temperature. 150 mg cloacal content or litter material wasdiluted in 1500 μl TBS (50 mM Tris, 150 mM NaCl, pH=7.2) with proteaseinhibitor cocktail (P2714, Sigma-Aldrich). The samples were mixed byvortex (2×1 min). Proteins (supernatants) were collected aftercentrifugation (13,000×g, 10′, 4° C.) and were used in duplicate ( 1/50dilution) in the ELISA (Chicken Ovotransferrin ELISA, KT-530, KamiyaBiomedical Company, Tukwila, USA). The ELISA was performed according tothe instructions of the manufacturer.

Statistical Analysis

Normality of the data was tested with the D'Agostino-Pearson normalitytest.

Differences in ovotransferrin levels between necrotic enteritis severitygroups (as measured by ELISA) were calculated using an a Kruskal-Wallistest, followed by a Dunn's post test.

Differences in ovotransferrin levels between the Eimeria-challenged andthe non-challenged control group were calculated using a Mann-Whitneytest.

The Spearman rank correlation was used to assess the relationshipbetween the ovotransferrin concentration in the cloacal samples andeither the necrotic enteritis lesion score or the coccidiosis score.Results were reported as means and standard error of the means (SEM).

Correlation of Fecal Ovotransferrin Concentration with Severity ofNecrotic Enteritis

Most samples from birds suffering from either mild necrotic enteritis(score 2) or without intestinal lesions (both challenged and negativecontrol animals) showed a low signal. In samples from birds with moresevere necrotic enteritis (necrosis score≥3), significantly moreovotransferrin was detected than in samples from challenged birds whodid not show intestinal disease (score 0), see Table 1. Furthermore,there was a positive correlation between the necrotic enteritis diseaseseverity and the ovotransferrin concentration in the fecal samples fromthe NE in vivo trial (p=0.0004).

neg. ctr (BHI) Score 0 Score 2 Score 3-4 Score 5-6 [μg/g] [μg/g] [μg/g][μg/g] [μg/g] Mean 1.80 1.01 1.16 3.59 5.87 Std. 0.4353 0.2605 0.19211.034 1.504 Error

Table 1 represents the ovotransferrin concentration (mean ± standarderror of the means) in faeces from birds that received all predisposingfactors but were not challenged with C. perfrirrgens (neg. ctr; n=8) orfrom birds challenged with C. perfringens resulting in varying degreesof necrotic enteritis: no necrotic lesions (score 0; n=8); mildintestinal necrosis (score 2; n=8); moderate necrotic enteritis (score3-4: n=8) or severe necrosis (score 5-6: n=8). * p<0.05.

Correlation of Fecal Ovotransferrin Concentration with Severity ofCoccidiose

Birds challenged with E. acervulina and E. tenella were used as a secondmodel for intestinal barrier failure. The ovotransferrin levels insamples from coccidiosis-positive birds were elevated as compared to theunchallenged controls (p=0.0029). Furthermore, there was a positivecorrelation between the coccidiosis score and the ovotransferrinconcentration in the faeces (p=0.0082). This difference inovotransferrin levels was also reflected in the litter samples, wheresignificantly higher ovotransferrin levels were detected in litter fromEimeria-challenged birds than in litter samples from non-challengedcontrol groups (p=0.0043), see Table 2.

neg. ctr coccidiosis Feces Litter Feces Litter [μg/g] [μg/g] [μg/g][μg/g] Mean 4.49 1.51 24.76 24.46 Std. 1.48 0.33 9.56 6.49 Error

Table 2 represents the ovotransferrin concentration (mean ± standarderror of the means) in faeces (grey) or mixed litter (white) fromexperimental coccidiosis-infected birds (coccidiosis; individual faecessamples: n=10 or mixed litter samples: n=6) or non-challenged controlbirds (neg. ctr; individual faeces: n=10 or mixed litter samples: n=5).Significant differences between the coccidiosis-positive group and thenon-challenged control group are indicated with ** p<0.01.

Results

As shown in the above, elevated fecal ovotransferrin levels weremeasured in birds with either experimental coccidiosis or necroticenteritis, which both cause intestinal barrier failure, using differentapproaches. ELISA analysis samples from different NE in vivo trialsrevealed that ovotransferrin was more abundant in samples from birdssuffering from necrotic enteritis as compared to unchallenged birds.Additionally, elevated ovotransferrin concentrations were measured insamples from coccidiosis-positive birds as compared to theirunchallenged controls.

Fecal ovotransferrin levels were significantly correlated with theseverity of intestinal barrier failure caused by either coccidiosis ornecrotic enteritis.

The degree of gut barrier failure might be classified depending on theseverity of the symptom on the affected sites (e.g. necrosis due to C.perfringens-induced necrotic enteritis), and the extent of the affectedsurface area. The degree of gut barrier failure is more severe with NEas this is associated with necrosis, the extent (in terms of surfacearea) is higher with coccidiosis.

As shown by the above experiments, the measurement of an specific APP(ovotransferrin) is a valuable tool to measure inflammation andconcomitant intestinal barrier failure, as it can provide information onspecific biological disease processes and is a useful tool to assessefficacy of molecules that reduce gastrointestinal disturbances.

1-14. (canceled)
 15. An in vitro method for detecting intestinal barrierfailure in animals, the method comprising the following steps: a)collecting intestinal sample material of an individual animal or of ananimal population; and b) determining the amount of at least one proteinmarker contained in said sample material; wherein: the at least oneprotein marker comprises ovotransferrin or a functional fragmentthereof; an increased amount of said at least one protein markercontained in said sample versus a reference sample indicates intestinalbarrier failure.
 16. The method of claim 15, wherein the referencesample is a species-specific control representing an intact intestinalbarrier.
 17. The method of claim 15, wherein the acute phase protein isdetected and quantified via an Enzyme-linked Immunosorbent Assay(ELISA).
 18. The method of claim 15, wherein the acute phase protein isdetected and quantified via a lateral flow assay.
 19. The method ofclaim 15, wherein the individual animal is an avian subject.
 20. Themethod of claim 15, wherein the animal population is an avianpopulation.
 21. The method of claim 15, wherein the intestinal samplematerial is a pooled sample.
 22. The method of claim 15, wherein theintestinal sample material is selected from the group consisting of gutcontent samples; samples of bodily excrements and solutions orsuspensions thereof; and materials being contaminated with bodilyexcrements.
 23. The method of claim 15, wherein the intestinal samplematerial is feces.
 24. The method of claim 15, wherein the intestinalsample material is a pooled fecal sample derived from an avianpopulation.
 25. The method of claim 15, wherein the intestinal samplematerial is stabilized immediately after sample collection.
 26. Themethod of claim 16, wherein the acute phase protein is detected andquantified via an Enzyme-linked Immunosorbent Assay (ELISA) or a lateralflow assay.
 27. The method of claim 26, wherein the individual animal isan avian subject.
 28. The method of claim 26, wherein the animalpopulation is an avian population, any


29. The method of claim 26, wherein the intestinal sample material is apooled sample, any


30. The method of claim 26, wherein the intestinal sample material isselected from the group consisting of gut content samples; samples ofbodily excrements and solutions or suspensions thereof; and materialsbeing contaminated with bodily excrements.
 31. The method of claim 26,wherein the intestinal sample material is feces or a pooled fecal samplederived from an avian population.
 32. The method of claim 31, whereinthe intestinal sample material is stabilized immediately after samplecollection.
 33. An in vitro method for detecting the extent ofintestinal barrier failure in animals, the method comprising thefollowing steps: a) collecting intestinal sample material of a specificanimal or of an animal population; and b) determining the amount of atleast one protein marker contained in the sample material; wherein: theat least one protein marker comprises ovotransferrin or a functionalfragment thereof; and the amount of said at least one protein markercontained in the sample indicates the extent of the intestinal barrierfailure.
 34. An in vitro method for monitoring the status of theintestinal barrier in animals, the method comprising the followingsteps: a) collecting intestinal sample material of a specific animal orof an animal population at consecutive points in time; b) determiningthe amount of at least one protein marker contained in the samplesobtained in step a); and c) determining deviations in the amounts ofsaid at least one protein marker contained in the samples obtained instep a); wherein the at least one protein marker comprisesovotransferrin or a functional fragment thereof.